Bottom Line:
Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins.An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed.Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF.

ABSTRACTAn effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.

gkt715-F6: Differential response of RPs to different levels of MMS damage. The response of two candidate induced RPs (Rpl7a-GFP, Rps22a-GFP) to MMS damage was investigated with live-cell flow cytometry, at 2 h. (A) At 0.02% MMS, the RPs are induced, whereas at the higher dose (0.1%), they are repressed. (B) Parental BY4741 cells were subjected to similar treatments, and culture densities (events/µl) were determined by flow cytometry (optical density measurements are expected to be not accurate at these early time points, as MMS arrest can cause an increase in cell-size). For three early log-phase cultures, initial densities were measured, and a third of each culture was subjected to 0, 0.02%, 0.1% MMS treatment. All densities at 2 h are normalized by the density of the initial cultures. 0.02% MMS significantly retards growth, but does not abrogate it altogether, whereas there is almost no growth in the presence of 0.1% MMS. (C) In a different assay from liquid cultures, even for cells growing on agar-pads with or without MMS, there is slow growth with 0.02% MMS, whereas no growth is seen at 0.1% MMS. Images of the same fields at 0 and 15 h are shown. (D) The GFP-tagged TF Sfp1, which controls RP gene expression, is induced at both doses of MMS as determined by live-cell flow cytometry. AF-corrected intensities are plotted; however, the expression of the protein is low in the absence of damage, making estimations of fold change difficult. (E) At 0.02% MMS, the protein is still nuclear, whereas at 0.1% MMS, it is clearly cytoplasmic, concomitant with the induction and repression of the RP proteins. Live cells were imaged with a 100× oil-immersion objective, given the low expression of the protein. Overlays of GFP and phase images are presented. The scalebar is 5 µm.

Mentions:
A surprising GO category for induced proteins was ribosome biogenesis and components of the ribosomal machinery because previous studies from several groups have shown that ribosomal genes are generally transcriptionally repressed under conditions of DNA damage (6,7,9,10). The regulation of ribosomal genes is thought to be primarily at the level of transcription in yeast (59), but almost 90% of all RP genes are also found in the list of PPT genes (13), suggesting cells use both transcription and translation to tune ribosome numbers (also see Supplementary Figure S16). A recent study similar to ours (discussed in more detail later in the text) also identified this group among induced proteins (20). To investigate this apparent discrepancy, we examined two such induced RPs (Rpl7a-GFP and Rps22a-GFP) using live-cell flow cytometry. We found that at moderate doses of MMS (0.02%, 2 h), both proteins were induced, whereas at higher MMS dose (0.1%, 2 h), there was a small but significant repression of the proteins (Figure 6A). The 2-h time-point was chosen because it was intermediate between the 3-h time-point at which the present screen is performed, and the 1-h time-point of the first transcriptional profiling study that revealed transcriptional repression of the ribosomal genes at 0.1% MMS (7). This suggests, not surprisingly, that cellular response differs according to the extent of damage, presumably depending on whether the cell can repair damage and proceed through the cell-cycle, or whether growth halts completely. A similar pattern of induction was seen in Tandem Affinity Purification (TAP)-tagged strains of Rpl7a and Rps22a (Supplementary Figure S18). Because ribosome biogenesis is intimately linked with cell growth, we investigated how cell growth characteristics differ at the two MMS doses.Figure 6.

gkt715-F6: Differential response of RPs to different levels of MMS damage. The response of two candidate induced RPs (Rpl7a-GFP, Rps22a-GFP) to MMS damage was investigated with live-cell flow cytometry, at 2 h. (A) At 0.02% MMS, the RPs are induced, whereas at the higher dose (0.1%), they are repressed. (B) Parental BY4741 cells were subjected to similar treatments, and culture densities (events/µl) were determined by flow cytometry (optical density measurements are expected to be not accurate at these early time points, as MMS arrest can cause an increase in cell-size). For three early log-phase cultures, initial densities were measured, and a third of each culture was subjected to 0, 0.02%, 0.1% MMS treatment. All densities at 2 h are normalized by the density of the initial cultures. 0.02% MMS significantly retards growth, but does not abrogate it altogether, whereas there is almost no growth in the presence of 0.1% MMS. (C) In a different assay from liquid cultures, even for cells growing on agar-pads with or without MMS, there is slow growth with 0.02% MMS, whereas no growth is seen at 0.1% MMS. Images of the same fields at 0 and 15 h are shown. (D) The GFP-tagged TF Sfp1, which controls RP gene expression, is induced at both doses of MMS as determined by live-cell flow cytometry. AF-corrected intensities are plotted; however, the expression of the protein is low in the absence of damage, making estimations of fold change difficult. (E) At 0.02% MMS, the protein is still nuclear, whereas at 0.1% MMS, it is clearly cytoplasmic, concomitant with the induction and repression of the RP proteins. Live cells were imaged with a 100× oil-immersion objective, given the low expression of the protein. Overlays of GFP and phase images are presented. The scalebar is 5 µm.

Mentions:
A surprising GO category for induced proteins was ribosome biogenesis and components of the ribosomal machinery because previous studies from several groups have shown that ribosomal genes are generally transcriptionally repressed under conditions of DNA damage (6,7,9,10). The regulation of ribosomal genes is thought to be primarily at the level of transcription in yeast (59), but almost 90% of all RP genes are also found in the list of PPT genes (13), suggesting cells use both transcription and translation to tune ribosome numbers (also see Supplementary Figure S16). A recent study similar to ours (discussed in more detail later in the text) also identified this group among induced proteins (20). To investigate this apparent discrepancy, we examined two such induced RPs (Rpl7a-GFP and Rps22a-GFP) using live-cell flow cytometry. We found that at moderate doses of MMS (0.02%, 2 h), both proteins were induced, whereas at higher MMS dose (0.1%, 2 h), there was a small but significant repression of the proteins (Figure 6A). The 2-h time-point was chosen because it was intermediate between the 3-h time-point at which the present screen is performed, and the 1-h time-point of the first transcriptional profiling study that revealed transcriptional repression of the ribosomal genes at 0.1% MMS (7). This suggests, not surprisingly, that cellular response differs according to the extent of damage, presumably depending on whether the cell can repair damage and proceed through the cell-cycle, or whether growth halts completely. A similar pattern of induction was seen in Tandem Affinity Purification (TAP)-tagged strains of Rpl7a and Rps22a (Supplementary Figure S18). Because ribosome biogenesis is intimately linked with cell growth, we investigated how cell growth characteristics differ at the two MMS doses.Figure 6.

Bottom Line:
Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins.An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed.Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF.

ABSTRACTAn effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.